Culturing adult rat hippocampal neurons with long-interval changing media

Background: Primary cultures of embryonic neurons have been used to introduce a model of neurons in physiological and pathological conditions. However, age-related cellular events limit this method as an optimal model in adult neurodegenerative diseases studies. Besides, short-interval changing media in previous cultures decreases the effectiveness of this model. As an example of this matter, we can refer to the study on some special neuronal secreted factors or the influence of some experimental materials on neurons. Meanwhile, short-interval changing media could remove the effects of some released factors from the environment. In this study, the method for isolation and culturing adult rat hippocampal neurons with long-intervals medium changing has been described. Methods: The hippocampal neurons of adult male rats were cultured. We used Neurobasal A/B27 culture medium, papain (2 mg/ml), trypsin 0.25% and collagenase (1 mg/ml) for neuronal isolation, OptiPrep density gradient for separation of neurons from other cell types and also debris and FGF2 (10 ng/ml) for increasing neuronal survival and regeneration. Results: The neuronal sprouting and viability were increased by using papain and mild triturating (P<0.05). Adult neuronal culturing and their regeneration were impossible without FGF2. It was shown that adding new fresh medium every 4 days and exchanging half of it every 8 days had no detrimental effect on neuronal viability. Conclusion: This investigation shows the possibility of culturing adult neuronal cells and their maintaining in long-interval media. It could be happened because of adult neurons rely significantly on the neighboring cells secreted factors for living and making synaptic connections. This model is very useful in physiological and pathological studies which need stable conditions of neuronal culture in a long period of time.     

Effects of different concentrations of bovine follicular fluid and estrous cow serum on development of murine 2-cell embryos

Murine 2-cells embryos were isolated from murine oviducts at laboratory and transferred into Ham's F-10 medium containing 0.1 mg mL(-1) streptomycin and 100 IU mL(-1) penicillin G and supplemented with 3 mg mL(-1) bovine serum albumin (BSA) or different concentrations of bovine follicular fluid (bFF) and estrous cow serum (ECS). Significantly higher (p<0.05) > or =4-cell embryos were developed when embryos were cultured 20% bFF (84.33%) comparing to 10 and 15% bFF (48.33 and 69.33%) as well as 3 mg mL(-1) BSA (65.66%). Morula rates were also lower in 10% bFF (22.33%) comparing to the other groups and were similar in 15 and 20% bFF (62.66 and 72.33% morula rates) as well as BSA containing media (55.33%). The highest (p<0.05) blastocyst rates were obtained in medium containing 20% bFF (64.33%) and the lowest belonged to 10% bFF (15%) comparing to 15% bFF (33.66%) or 3 mg mL(-1) BSA. When embryos were cultured in ECS, no significant different was observed in different culture media (76.66, 72.33, 82.5 and 65.66% > or =4-cell embryos in 10, 15 and 20% bFF and 3 mg mL(-1) BSA, respectively). Morula and blastocyst rates were also similar in all groups (32.33, 41.66 and 66.25 and 55.33% morula rates and 15.33, 27, 44.50 and 29.66% blastocyst rates for 10, 15 and 20% bFF and 3 mg mL(-1) BSA, respectively). The results of the present study demonstrated that 20% bFF could be substituted for BSA when in vitro culture of murine embryos is carried.     

Isolation of Streptococci from milk samples of normal, acute and subclinical mastitis cows and determination of their antibiotic susceptibility patterns

Streptococci are frequently isolated from bovine mastitis in dairy cows with only limited information available on the antimicrobial susceptibility of these organisms. A total of 42 Streptococci isolated from 148 milk samples of normal, sub acute and acute bovine mastitis cases. Overall, 35% of the strains tested were Streptococcus dysgalactiae, Streptococcus agalactiae 26%, Streptococcus uberis 18 and 4% were Enterococcus sp. Differences between the number of isolations in acute and sub acute groups were statistically significant, (p<0.5). The antimicrobial susceptibility for these organisms was determined for the following antimicrobial agents: cephalexine, penicillin, clindamycin, cloxaciline, gentamicin, streptomycin, amoxicillin, tetracycline, kanamycin, oxytetracycline, ampicillin, chloramphenicol and erythromycin. S. agalactiae, S. dysgalactiae, S. uberis and Enterococci demonstrated high level of resistance against streptomycin, penicillin and cloxaciline. Low level of sensitivity to other tested antimicrobials was demonstrated.     

Immense essence of excellence: marine microbial bioactive compounds

Oceans have borne most of the biological activities on our planet. A number of biologically active compounds with varying degrees of action, such as anti-tumor, anti-cancer, anti-microtubule, anti-proliferative, cytotoxic, photo protective, as well as antibiotic and antifouling properties, have been isolated to date from marine sources. The marine environment also represents a largely unexplored source for isolation of new microbes (bacteria, fungi, actinomycetes, microalgae-cyanobacteria and diatoms) that are potent producers of bioactive secondary metabolites. Extensive research has been done to unveil the bioactive potential of marine microbes (free living and symbiotic) and the results are amazingly diverse and productive. Some of these bioactive secondary metabolites of microbial origin with strong antibacterial and antifungal activities are being intensely used as antibiotics and may be effective against infectious diseases such as HIV, conditions of multiple bacterial infections (penicillin, cephalosporines, streptomycin, and vancomycin) or neuropsychiatric sequelae. Research is also being conducted on the general aspects of biophysical and biochemical properties, chemical structures and biotechnological applications of the bioactive substances derived from marine microorganisms, and their potential use as cosmeceuticals and nutraceuticals. This review is an attempt to consolidate the latest studies and critical research in this field, and to showcase the immense competence of marine microbial flora as bioactive metabolite producers. In addition, the present review addresses some effective and novel approaches of procuring marine microbial compounds utilizing the latest screening strategies of drug discovery.     

Delivery of epidermal neural crest stem cells (EPI-NCSC) to hippocamp in Alzheimer's disease rat model

Background: Alzheimer’s disease (AD) is characterized by progressive neuronal loss in hippocamp. Epidermal neural crest stem cells (EPI-NCSC) can differentiate into neurons, astrocytes and oligodendrocytes. The purpose of this study was to evaluate the effects of transplanting EPI-NCSC into AD rat model. Methods: Two weeks after induction of AD by injection of Amyloid-β 1-40 into CA1 area of rat hippocamp, Y-maze and single-trial passive avoidance tests were used to show deficit of learning and memory abilities. EPI-NCSC were obtained from the vibrissa hair follicle of rat, cultured and labeled with bromodeoxyuridine. When Alzheimer was proved by behavioral tests, EPI-NCSC was transplanted into CA3 area of hippocamp in AD rat model. The staining of EPI-NCSC markers (nestin and SOX10) was done in vitro. Double-labeling immunofluorescence was performed to study survival and differentiation of the grafted cells. Results: We showed that transplanted EPI-NCSC survive and produce many neurons and a few glial cells, presenting glial fibrillary acidic protein. Total number of granule cells in hippocamp was estimated to be more in the AD rat model with transplanted cells as compared to AD control group. We observed that rats with hippocampal damage made more errors than control rats on the Y-maze, when reward locations were reversed. Conclusion: Transplanted cells were migrated to all areas of hippocamp and the total number of granule cell in treatment group was equal compared to control group. Transplantation of EPI-NCSC into hippocamp might differentiate into cholinergic neurons and could cure impairment of memory in AD rat model.Key Words: Alzheimer’s disease, Cholinergic neuron, Hair follicle     

The expression of signal regulatory protein-alpha in normal and osteoarthritic human articular cartilage and its involvement in chondrocyte mechano-transduction response

BACKGROUND: Signal regulatory proteins (SIRP) belong to immunoglobulin super family (IgSF) and relate to integrin signaling cascades. It has been shown that SIRPalpha is expressed in a variety of cells including myeloid cells and neurons. In the present study the expression of this IgSF member in articular chondrocytes was investigated. METHODS: Using a panel of anti-SIRPalpha antibodies, immunohistochemistry, Western-blotting and electrophysiology methods, expression of SIRPalpha and its role in chondrocyte mechano-transduction were assessed. RESULTS: No identifiable positive signal was obtained by using immunohistochemistry methods on frozen and paraffin sections. SIRPalpha is expressed by both normal and osteoarthritis cultured chondrocytes. The electrophysiological response of chondrocytes in the presence of SE7C2 mAb was significantly inhibited whereas; SE5A5 did not show any modification in this response. CONCLUSIONS: It seems likely that SIRPalpha could be associated with other proteins such as integrins, CD47 and ion channels, which contribute to the electrophysiological response of human articular chondrocytes. In any case, this study has provided a specific functional role for SIRPalpha in chondrocyte mechano-transduction.     

椰乳在甘蔗组培快繁中作用的研究

以种性优良但组培分化斟难的3个甘蔗品种FN95-1702、FN99-2308和FN99-20169的愈伤组织为材料,以2种常用的培养基为基本培养基,分别添加4种不同体积的椰乳,采用随机区组实验设计,进行分化阶段和生根阶段培养,并对移栽幼苗成活率进行了观察.结果表明:对于不同激素配方的培养基,在分化阶段添加椰乳后甘蔗愈伤组织的分化率和芽长势均明显提高,添加椰乳的适宜体积为10%～20%,其中,以15%为最佳添加体积,可使分化率提高100%.幼苗生根阶段,添加椰乳使幼苗生根的数量减少,对移栽成活率影响不明显.不同甘蔗品种适合的培养基略有差异,实验所选用的2种培养基与椰乳之间无交互作用.     

马铃薯脱毒试管苗快繁中污染抑菌剂的筛选

在马铃薯脱毒试管苗工厂化快繁过程中,常因各种原因出现细菌污染,严重影响试管苗的生长,给生产带来损失。为了解决这一问题,本试验分别加入卡那霉素、青霉素、链霉素及青霉素和链霉素配合使用的抗生素,研究抑菌剂及抑菌剂浓度在马铃薯快繁过程中的抑制细菌的效果。结果表明:在培养基中加入卡那霉素、青霉素G钠、青霉素和链霉素的混合物都能抑制细菌的污染,但青霉素各处理不仅能抑制细菌污染,而且对试管苗的生长有明显的促进作用,其中青霉素60 mg.L-1的效果最好,同时抑菌剂的抑菌作用具有时效性。     

Growth of primary hippocampal neurons on multichannel silk fibroin scaffold

Particular attention has been given to axonal outgrowth of neurons to understand how topographical surface cues influence attachment and subsequent directional migration and growth. In present study, the silk fibroin (SF) scaffold with uniaxial channels was prepared by directional freeze-drying processes. The average pore diameter, the porosity, and pore density of the scaffold are 120 µm, 88 %, and 203 mm^?2, respectively. Further, hippocampal neurons were seeded onto the scaffold and the hippocampal neurons morphology was investigated. Cell-cell networks and cell-matrix interactions had been established by newly formed axons and the diversity of neurons was much higher after culturing 7 days. The neurons expressed β-III-tubulin and nerve filament, while glial fibrillary acidic protein immunofluorescence was barely above background. These results indicated that the SF scaffolds with uniaxial multichannels could be guided axons of neurons spread along the channels. SF scaffolds with oriented pores have a potential for nerve tissue regeneration.     

Effects of Various Carbon Sources and Their Combinations on in vitro Growth and Photosynthesis of Banana Plantlets

abstract

The effects of various carbon sources, sucrose, glucose and fructose alone or in combination on the in vitro growth of banana plantlets were studied. Banana plants were cultured on the media supplemented with these carbon sources at 0.08 M for 13 weeks. The water potential of the medium was the highest in the medium supplemented with sucrose + glucose (-0.3 MPa), and was significantly lower in the medium supplemented with fructose alone or in combination with other carbon sources (-0.7 to -1.0 MPa) than in the other media. The leaf water potential was also the highest in the plants cultured on the medium supplemented with sucrose + glucose, and lowest in the plants cultured on that with fructose. The leaf water potential of plants cultured on sucrose + glucose, sucrose and glucose correlated well with their growth and photosynthetic activity, but the correlation was not observed in the plants cultured on fructose alone or in combination with other carbon sources. Plants cultured on fructose had a lower chlorophyll content (400 ptg dm^-2) and lower photosynthetic rate (3 μmol02 m^-2 s^-1) than those cultured on sucrose + glucose (15,950 μgdm^-2 for chlorophyll and 8.5 μmol02 m^-2 s^-1 for photosynthesis), and these differences were statistically significant. Both chlorophyll content and photosynthetic oxygen evolution were the highest in the plants cultured on sucrose + glucose, and the superior growth of plants on this medium was attributed to their high photosynthetic efficiency.     

The effect of 2,4-d on tuberization and starch content of potato tubers produced on stem segments culturedin vitro

The effect of 2,4-D on tuberization, yield and the starch content of potato tubers produced on stem segments was examined. Potato stem segments were cultured in nutrient agar containing 2,4-D concentrations ranging from 10^?7 M to 2 × 10^?3 M. The stem segments were incubated in the dark at 25° C and examined after 15 days. The degree of tuberization on stem segments cultured in media containing 10^?7 M and 10^?6 M 2,4-D was higher than on the control stem segments. Mainly long stolons were observed on stem segments cultured in media containing 10^?4 M and 2 × 10^?3 M 2,4-D. The percent starch (dry wt. basis) in stolons was higher than that in tubers. However, the total starch content of tubers per stem segment cultured in media containing 10^?7 M and 10^?6 M 2,4-D was significantly higher than that of stolons obtained from stem segments cultured in media containing higher concentrations of 2,4-D. The total starch content of tubers, except those produced on stem segments cultured in media containing 10^?7 M 2,4-D, decreased steadily with increasing concentration of 2,4-D in the culture media.     

Effects of different organic additives on in vitro shoot regeneration of Celosia sp

Nowadays, many researches were conducted in minimizing tissue culture technology due to the overhead of cost needed. The purpose of this study was to investigate the effects of using five kinds of organic additives at four level concentrations responsive to the number of shoots produced for eight weeks in culture. Stem segment explants of Celosia sp. were cultured on MS medium that have been supplemented with different kinds of extract juice that serve as organic additives which are mature coconut, young coconut, papaya, banana and tomato at 20, 30, 50 and 70 ml L-1. The numbers of shoot on each explant were recorded and the mean of ten replicates explants were calculated. Among the media used, young coconut water at 70 ml L1- induced the highest shoot regeneration (14.21+/-8.26), followed by mature coconut water at 50 ml L-1 (13.14+/-10.33). Banana and tomato juice promote highest shoot regeneration of stem segments at 50 ml L-1 that produced 9.57+/-4.68 and 9.28+/-5.82 shoots per explants, respectively. While the lowest concentration which at 20 ml L-1 of papaya juice showed highest shoot regeneration (10.50+/-3.45) produced among the three other concentration tested. Statistical results showed that there were significant differences interactions effects (p<0.05) in terms of number of shoot regenerated between the types of extracts juices determined by ANOVA test. Comparing number of shoots regenerated that were cultured in control media, it showed higher than all of experimental medium composition. There were no big different in cost required in preparation of control media and the experimental media. Applications of five kinds of local fruit in tissue culture media should be considered since it responsive in shoot regeneration.     

影响柑桔球形合子胚离体培养的若干因素

通过对柑桔球形合子胚不同发育期的分离培养、不同基本培养基及附加物、蔗糖浓度等因素对柑桔合子胚离体培养影响的研,结果表明:授粉后50d是柑桔合子胚离体培养的最适时期;Whiet基本培养基比MS基本培养基更适于柑桔球形合子胚离体培养。适当增加培养基的渗透压和添加水解乳蛋白,特别是袖子胚乳 ,可提高幼胚的诱导率,有利于柑桔球形胚的发育。比较适宜的蔗糖浓度为10%一 15%,水解乳蛋白浓度为400mg/L     

Surface coating as a key parameter in engineering neuronal network structures in vitro

By quantitatively comparing a variety of macromolecular surface coating agents, we discovered that surface coating strongly modulates the adhesion and morphogenesis of primary hippocampal neurons and serves as a switch of somata clustering and neurite fasciculation in vitro. The kinetics of neuronal adhesion on poly-lysine-coated surfaces is much faster than that on laminin and Matrigel-coated surfaces, and the distribution of adhesion is more homogenous on poly-lysine. Matrigel and laminin, on the other hand, facilitate neuritogenesis more than poly-lysine does. Eventually, on Matrigel-coated surfaces of self-assembled monolayers, neurons tend to undergo somata clustering and neurite fasciculation. By replacing coating proteins with cerebral astrocytes, and patterning neurons on astrocytes through self-assembled monolayers, microfluidics and micro-contact printing, we found that astrocyte promotes soma adhesion and astrocyte processes guide neurites. There, astrocytes could be a versatile substrate in engineering neuronal networks in vitro. Besides, quantitative measurements of cellular responses on various coatings would be valuable information for the neurobiology community in the choice of the most appropriate coating strategy.     

复方刺五加注射液对培养大鼠心肌细胞动作电位的影响

培养Wistar大鼠乳鼠心室肌细胞,向培养基中加入复方刺五加注射液200μg/mL,可使心肌细胞动作电位的波幅、波宽、阈电位、最大舒张电位、最大除极速度及复极(10%、50%、90%)水平的动作电位波宽一致减小,其作用与钙通道阻滞剂尼莫地平0.4μg/mL相似;Ca2 80μg/mL能使上述作用反转,提示复方刺五加注射液具有钙通道阻滞作用。     

促生细菌Bacillus sp. QN2MO-1的鉴定及对番茄生长的影响

为分离和鉴定具有促生作用的内生菌,开发潜在功能的微生物菌株,本研究以诺尼（Morinda citrifolia L.）为研究对象,从植株不同组织中共分离得到621株内生细菌,经复筛得到具有促生和解磷作用的菌株,命名为QN₂MO-1。基于菌株的生理生化分析和16S rDNA的序列比对,该内生细菌与Bacillus siamensis KCTC 13613^T（AJVF01000043）具有99.04%的同源性。药敏性分析显示,该菌对青霉素、硫酸链霉素和氨苄西林具有明显的抗性。QN₂MO-1可促进番茄种子的萌发;与对照相比,5%菌体发酵液处理能显著提高植株的生长。     

芸薹属远缘杂交试管苗快繁培养基的优化

为了优化芸薹属远缘杂交试管苗的快繁培养基,探讨基因型、培养时间等因素对试管苗分化率的影响,实验对甘蓝型油菜与甘蓝以及白菜型油菜与甘蓝两种远缘杂交方式的试管苗,在10种添加了不同浓度的6-BA和NAA的培养基上进行快繁培养。采用SAS软件对培养后第20d和第30d的分化率进行分析处理,结果如下：（1）白菜型油菜与甘蓝5个杂交组合间,以及白菜型油菜与甘蓝、甘蓝型油菜与甘蓝两种杂交类型间的试管苗分化率无显著差异;除甘蓝型油菜与甘蓝杂交的试管苗在培养第30d时的分化率无显著差异外,其它情况下培养基间的诱导分化率差异均达到显著或极显著水平;（2）添加3.0 mg&;#8226;L-1 6-BA（6-苄基嘌呤）和0.2 mg&;#8226;L-1 NAA（萘乙酸）的MS培养基对试管苗的诱导分化效率显著或极显著高于其它快繁培养基;（3）不同培养基培养的试管苗在培养20d和30d时的分化率呈高度正相关,但前20d的日平均分化率显著高于20~30d的日平均分化率。     

Comparison of different culture media on the mycelial growth, sporangia and oospore production ofPhytophthora infestans

The purpose of this study was to evaluate various culture media, with or without β-sitosterol, on mycelial growth, sporangia and oospore production by isolates ofPhytophthora infestans. Nine media were compared to rye agar (RA) which is frequently used to cultureP. infestans. Poor fungal growth across most isolates was observed on soybean agar, carrot agar and carrot agar with sterols relative to RA. Only clarified V8 juice containing β-sitosterol (CLV8S) resulted in significantly more sporangia production compared to RA. All other media performed similar to the RA. No significant differences were found in the sizes (length and width) of sporangia produced among the isolates tested on various media. Oospore production was greatest whenP. infestans was cultured on carrot agar with β-sitosterol (CAS) or CLV8S when compared to RA. Responses of isolate matings varied somewhat with an A2 isolate from Nova Scotia consistently producing fewer oospores in matings with A1 isolates compared to the other A1/A2 mating combinations. Rye agar and oat meal agar with or without β-sitosterol as well as clarified V8 juice agar with β-sitosterol are useful forin vitro growth of mycelium.     

金花茶松散型愈伤组织诱导与悬浮细胞系建立

采用组织培养及显微观察技术,以金花茶植株茎段、叶片、花药及离体培养的金花茶体细胞胚为试验材料,对松散型愈伤组织的获得及悬浮细胞系的建立进行了研究。结果表明:在黑暗条件下,以金花茶体细胞胚切块为外植体,在MS+KT 0.5 mg/L+NAA 8.0 mg/L+硝酸银5mg/L+蔗糖30 g/L培养基上诱导出愈伤组织后,将其转接到该诱导培养基上连续培养3个月,最后从培养物中挑选状态良好的松散型愈伤组织在分别含有肌醇与硝酸银的2种培养基上交替培养,可以获得金花茶松散型愈伤组织;方差分析表明:蔗糖添加量、肌醇添加量及硝酸银添加量均对金花茶悬浮培养液细胞密度有显著影响且后两者分别与光照条件存在相互作用;将松散型愈伤组织在分别添加100 mg/L肌醇与2.5 mg/L硝酸银的液体增殖培养基上交替培养,可以建立并保持金花茶悬浮细胞系。该研究结果可为金花茶大规模细胞培养提供科学依据。     

Comparison of distribution of virulence determinants in clinical and environmental isolates of Vibrio cholera

Background: The virulence of a pathogenic Vibrio cholerae is dependent on a discrete set of genetic determinants. In this study, we determined the distribution of virulence determinants among the clinical and environmental isolates of V. cholerae. Methods: The antibiotic resistance profiles of the isolates were determined using standard disk diffusion assay. PCR assay was performed to analyze the presence of toxin genes of ctx, zot and ace. The composition of cholera toxin encoding element (CTX) region flanking of the V. cholerae isolates was also analyzed. Results: All of the clinical isolates (100%) showed a complete set of virulence genes and also the attachment site of the filamentous bacteriophage CTXphi. None of the environmental isolates contained the virulence genes and the attachment site of the CTXphi. Analysis of the flanking regions including the toxin-linked cryptic element and repeat in toxin genes revealed their integrity in the clinical isolates while in the environmental isolates they were absent or contained incomplete sequences. Comparison of the antibiotic resistance assay of the environmental and clinical isolates showed a significant difference in the resistance profiles of the isolates obtained from the two sites. High rates of resistance to co-trimoxosol, streptomycin and chloramphenicol were found with clinical isolates. Conclusion: The absence of all virulence determinants in the environmental strains may suggest that certain ecological features must be present for V. cholerae to acquire a complete set of virulence determinants and to turn them into pathogenic strains.